Jurkat project

Project description

We are studying the effect of an inhibitor of the cGAS-STING signaling pathway H151 on T-ALL model cell line Jurkat.

The biological experiment revealed that H151 causes cell death, so the pathway is important for survival of T-ALL.

We want to explore the differential expressed genes between normal condition and by inhibiting the pathway. Thus we are going to perform a differential expression analysis (DEA) followed by a pathway enrichment analysis (PEA)

Following a basic RNA-seq pipeline analysis

Importing data

Raw counts

Metadata

Experimental data

Data exploration

First let’s view the distribution of the different bio types we have in our data :

In the downstream analysis (DEA), we’ll be focusing on the top 2 biotypes (protein_coding and lncRNA). Additional filtering will be applied :

• MaxCount_threshold = 20 (At least 1 sample must have a read count over that value)
• CpmCount_threshold = 0.5 (Count per million reads threshold)
• MinSample = 3 (Samples that should pass the cpm threshold)

Now to understand the global gene expression landscape and to assess the quality control of our data, we need to perform a dimentionality reduction analysis : Principal component analysis (PCA)

The PCA :

• Assumes homoscedasticity (equal variance) between the samples
• Assumes roughly normal distribution

So we need to perform some data transformation first on raw counts.

We’ll be using a Variance Stabilizing Transformation (VST) from the DESeq2 package. And This will :

• Normalize for library size (DESeq2 computes size factors using a median-of-ratios method)
• Use a negative binomial model to estimate dispersion
• Apply a variance-stabilizing mathematical transformation

Differential expression analysis (DEA)

Now let’s dive into gene expression.

dds <- pre_process_results$DESeqData
dds$Condition <- relevel(dds$Condition, ref = "Jurkat_ct") ## set the control
dds <- DESeq(dds) 

Let’s get a quick overview of the distribution of the DEGs

• Volcano plot:

• Heatmap:

DEGs list overview

• Up regulated genes: 261

##   [1] "MIR210HG"        "ENSG00000288930" "CXCR3"           "APLN"           
##   [5] "H2BC21"          "RANBP3-DT"       "RGS6"            "IRAIN"          
##   [9] "ENSG00000196465" "BHLHE40"         "ENSG00000181577" "ENSG00000146223"
##  [13] "COL6A3"          "LINC00484"       "ENSG00000125651" "VGF"            
##  [17] "SLAMF8"          "KANK4"           "ENSG00000211454" "WNT8B"          
##  [21] "TMEM240"         "ENSG00000236204" "MSLNL"           "ENSG00000213139"
##  [25] "TMEM163"         "NANOS1"          "LINC01963"       "ENSG00000170571"
##  [29] "RPRML"           "ENSG00000086300" "ENSG00000136840" "CCR8"           
##  [33] "ENSG00000290124" "KIT"             "ENSG00000269968" "ENSG00000230148"
##  [37] "TMEM269"         "ILF3-DT"         "ENSG00000173914" "TERT"           
##  [41] "CLEC12A"         "ENSG00000171357" "ENSG00000138614" "PKD1P6-NPIPP1"  
##  [45] "RNASEH2A"        "DTL"             "ENSG00000261226" "UNG"            
##  [49] "ENSG00000183979" "ENSG00000165028" "ENSG00000182197" "ASH1L-AS1"      
##  [53] "ENSG00000205903" "MCM4"            "TOLLIP-DT"       "MIF4GD-DT"      
##  [57] "ENSG00000272356" "ZNF674-AS1"      "ENSG00000198034" "TCHH"           
##  [61] "LVRN"            "ENSG00000143971" "YTHDF3-DT"       "ENSG00000287362"
##  [65] "ENSG00000277767" "GPR146"          "ENSG00000289128" "FAM30A"         
##  [69] "ENSG00000233178" "ENSG00000276390" "ENSG00000187164" "ENSG00000142556"
##  [73] "CXXC4"           "ENSG00000124370" "REEP1"           "LRRC10B"        
##  [77] "DDIT4"           "ENSG00000167863" "H4C9"            "ENSG00000224066"
##  [81] "ENSG00000151093" "ENSG00000165475" "C16orf95-DT"     "TINCR"          
##  [85] "ENSG00000289985" "ZNF346-IT1"      "DDT"             "ENSG00000259736"
##  [89] "SCLY"            "ENSG00000288586" "HERC3"           "PTPN6"          
##  [93] "ENSG00000278611" "SLC37A2"         "ENSG00000140092" "ENSG00000231527"
##  [97] "TRARG1"          "DENND2D"         "TMEM255B"        "FOXD2-AS1"      
## [101] "LINC00641"       "LRRC8D-DT"       "NIPBL-DT"        "ENSG00000138738"
## [105] "PCNA"            "ENSG00000251247" "ENSG00000183891" "ENSG00000163935"
## [109] "GET1"            "MYT1L"           "UHRF1"           "C1S"            
## [113] "PRKCZ-AS1"       "SLFNL1-AS1"      "ENSG00000137941" "SLC16A4-AS1"    
## [117] "ELFN2"           "FAM81A"          "SASH3"           "ZNF710-AS1"     
## [121] "JAKMIP1"         "MAD2L1-DT"       "ENSG00000260442" "TWIST1"         
## [125] "ABTB3"           "ENSG00000244184" "ADGRL2"          "GJD3-AS1"       
## [129] "MYO18B"          "TBC1D22A-DT"     "ENSG00000157800" "ENSG00000126107"
## [133] "ENSG00000102409" "MHENCR"          "CSPG5"           "LNP1"           
## [137] "GNA15"           "ENSG00000197548" "ENSG00000133794" "MCM2"           
## [141] "SNHG10"          "P2RX6"           "FA2H"            "MCM3"           
## [145] "C9orf163"        "ENSG00000175040" "OPN3"            "ENSG00000143740"
## [149] "SLBP"            "NECAB3"          "ENSG00000105808" "RASSF1-AS1"     
## [153] "ENSG00000178982" "ENSG00000185621" "PDK1-AS1"        "ENSG00000291056"
## [157] "SUGT1-DT"        "ENSG00000124140" "LINC01311"       "ENSG00000285437"
## [161] "EID2B"           "ENSG00000100003" "PDE4A"           "MCM7"           
## [165] "NKILA"           "IGLL1"           "ENSG00000243479" "ENSG00000100321"
## [169] "NOTCH1"          "ENSG00000287104" "ENSG00000158555" "PPM1H"          
## [173] "ENSG00000153936" "PDXP"            "MCPH1-DT"        "RNF139-DT"      
## [177] "ENSG00000173218" "ENSG00000155366" "ENSG00000267757" "ENSG00000055950"
## [181] "ENSG00000162413" "JMJD7"           "MCF2L-AS1"       "ENSG00000246308"
## [185] "ENSG00000065413" "DTX1"            "CALML4"          "FEN1"           
## [189] "ENSG00000113595" "RMI2"            "LINC01284"       "ENSG00000139266"
## [193] "KRT72"           "HK2"             "ENSG00000153561" "GNMT"           
## [197] "ENSG00000118242" "GABPB1-IT1"      "TCF7"            "PIGW"           
## [201] "CIRBP-AS1"       "ATP2B2"          "IMPDH2"          "ZNF704"         
## [205] "ENSG00000267688" "ENSG00000114019" "H3C10"           "LINC01971"      
## [209] "OXTR"            "ENSG00000068394" "ENSG00000185885" "PARTICL"        
## [213] "PANO1"           "LINC00235"       "MIR193BHG"       "FBXL22"         
## [217] "ST3GAL1-DT"      "ENSG00000196810" "TTC7A"           "NT5DC2"         
## [221] "POLG"            "ENSG00000286548" "ENSG00000006194" "NPIPB13"        
## [225] "PAXIP1-DT"       "ANKRD37"         "NUP210"          "ENSG00000106336"
## [229] "LINC00528"       "CNN3-DT"         "WT1"             "ENSG00000198353"
## [233] "ZMYND19"         "ENSG00000108479" "PRKCH-AS1"       "ENSG00000221823"
## [237] "ENSG00000165915" "ENSG00000160867" "LINC02019"       "YEATS2-AS1"     
## [241] "RHPN2"           "CXXC5"           "NCBP2AS2"        "STAM-DT"        
## [245] "ENSG00000104219" "LINC00304"       "NPIPB11"         "RORB-AS1"       
## [249] "ENSG00000149823" "LINC01422"       "ENSG00000177556" "GNG4"           
## [253] "DNAH10OS"        "NOTCH2NLB"       "LINC01389"       "ENSG00000101336"
## [257] "ENSG00000276853" "ENSG00000213047" "LINC02918"       "DANCR"          
## [261] "IVNS1ABP"

• Down regulated genes: 650

##   [1] "CEP126"          "ZNF836"          "SLC26A11"        "CLEC18B"        
##   [5] "PCDHGA6"         "SLC8A2"          "PLCD1"           "RNFT1"          
##   [9] "RAP2C-AS1"       "RELB"            "DDX19A-DT"       "ZBTB7B"         
##  [13] "SP2-DT"          "ANKRD31"         "PCYT1B"          "ENSG00000204482"
##  [17] "LINC01058"       "MACO1"           "LINC00652"       "LINC01134"      
##  [21] "LINC01215"       "LINC-PINT"       "USP27X-DT"       "NQO2"           
##  [25] "NIPAL1"          "PLTP"            "ZNF585B"         "NPIPA5"         
##  [29] "STPG1"           "ENSG00000289958" "RPAP3-DT"        "DMGDH"          
##  [33] "CFAP69"          "ZNF329"          "APOBEC3G"        "CLIC4"          
##  [37] "C2orf50"         "ENSG00000138061" "SERPINI1"        "ENSG00000272438"
##  [41] "B3GNT5"          "PLEKHA6"         "GDPD1"           "MAP1A"          
##  [45] "GABRB2"          "DUSP16"          "ERCC6L2-AS1"     "CCT6B"          
##  [49] "GARIN1A"         "ACTRT3"          "ELL2"            "BCHE"           
##  [53] "SHF"             "TCP11L2"         "ARMH4"           "TMEM79"         
##  [57] "TSPAN10"         "PCAT1"           "ENSG00000102245" "PEX5L"          
##  [61] "SNRPA1-DT"       "ACTR3-AS1"       "MYO5B"           "TBC1D30"        
##  [65] "FGF22"           "ENSG00000261173" "LINC01115"       "RGPD3"          
##  [69] "ANG"             "SPRY3"           "IFI35"           "CPEB1-AS1"      
##  [73] "DOK4"            "ENSG00000177788" "IFRD1"           "PRDM8-AS1"      
##  [77] "MIR4280HG"       "CASP9"           "LINC00662"       "CNGA1"          
##  [81] "SPTB"            "ZNF610"          "C3AR1"           "ENSG00000291010"
##  [85] "PCDHGA10"        "ERRFI1"          "TWF2-DT"         "FRRS1"          
##  [89] "TNS2"            "MPZL3"           "TSNAXIP1"        "EGFL7"          
##  [93] "RORA-AS1"        "ENSG00000178896" "SCG2"            "COPB2-DT"       
##  [97] "ZNF225-AS1"      "SLC16A9"         "DENND2C"         "RSPH4A"         
## [101] "ENSG00000197024" "LNCRNA-IUR"      "LINC01465"       "SLC17A7"        
## [105] "FZD7"            "MCTP1"           "HOXB6"           "WNK4"           
## [109] "RAB39B"          "NUDT13"          "LRP12"           "LINC00853"      
## [113] "CYP2J2"          "WDR31"           "MIR4435-2HG"     "CTSG"           
## [117] "ENSG00000105698" "ENSG00000213588" "ID1"             "ENSG00000213465"
## [121] "SLCO3A1"         "SYNE4"           "CD84"            "SLC38A3"        
## [125] "FAM219A"         "CYSRT1"          "SERTAD1"         "DNAAF8"         
## [129] "LAT2"            "CA13"            "DNAJC3"          "TNN"            
## [133] "ERBB2"           "CAPS"            "JAM2"            "ULBP2"          
## [137] "RXRA"            "FAM174A-DT"      "FLVCR2"          "ZNF451-AS1"     
## [141] "LINC02709"       "PILRA"           "C11orf65"        "HDAC2-AS2"      
## [145] "SPNS3"           "ENSG00000260874" "ICOS"            "CBS"            
## [149] "ENSG00000125775" "PCOTH"           "EVI2A"           "RTN2"           
## [153] "ADAM8"           "IL20RB"          "BAZ2B-AS1"       "BEND6"          
## [157] "FTH1"            "TMEM151A"        "TMEM267"         "GCNT4"          
## [161] "PEX11G"          "C19orf38"        "BTG1-DT"         "DNAI7"          
## [165] "DDRGK1"          "CCDC184"         "P2RY10"          "ENSG00000228436"
## [169] "SDCBP2-AS1"      "TMBIM1"          "ENSG00000140104" "OASL"           
## [173] "FOSB"            "DBNDD1"          "CCDC69"          "NECTIN3"        
## [177] "ENSG00000108599" "JAML"            "ENSG00000275793" "TMEM254-AS1"    
## [181] "ENSG00000285706" "IRAK2"           "EFNB2"           "ENSG00000288996"
## [185] "TAGLN"           "HULC"            "BVES"            "SYNGR4"         
## [189] "CACNG8"          "ALG1L9P"         "CCNB3"           "FMO4"           
## [193] "PEAR1"           "SLC43A1"         "MIA2"            "ZFAND2A-DT"     
## [197] "RAB17"           "LINC01825"       "CDS1"            "EVA1B"          
## [201] "TPST1"           "RPL37A-DT"       "CYB5R1"          "C14orf39"       
## [205] "NES"             "APOL1"           "SSC4D"           "TXNRD1"         
## [209] "C22orf23"        "METTL27"         "EHD2"            "SAMD12-AS1"     
## [213] "PDGFRL"          "MAML2"           "ZNF235"          "GAB2"           
## [217] "CGRRF1"          "DAPP1"           "LCA5L"           "ENSG00000162543"
## [221] "DNAJB9"          "IRAG2"           "CCDC65"          "GPT2"           
## [225] "SLC9A9"          "LINC00877"       "WIPI1"           "EPHX1"          
## [229] "LYPLAL1-DT"      "MN1"             "MKLN1-AS"        "BTG2-DT"        
## [233] "ENSG00000188761" "SEL1L3"          "ENSG00000283141" "NUTM2E"         
## [237] "RBP5"            "BBC3"            "EPS8"            "NEK5"           
## [241] "ASS1"            "ECE1-AS1"        "ARHGAP29"        "RGS17"          
## [245] "EIF4EBP1"        "ABHD3"           "TXK"             "EID3"           
## [249] "DLGAP1-AS2"      "CTSO"            "WEE2-AS1"        "ENSG00000021574"
## [253] "PGGHG"           "ACTN2"           "BMP10"           "OXR1-AS1"       
## [257] "KIF5C"           "CYP1A1"          "MFAP3L"          "LINC00680"      
## [261] "TRIM22"          "LAMC3"           "ZFP2"            "ENSG00000289183"
## [265] "ZNF582"          "ENSG00000273183" "PCLO"            "PLEKHH3"        
## [269] "SPOPL-DT"        "EAF2"            "LINC02901"       "LGALS8-AS1"     
## [273] "NAGS"            "ATF3"            "HSPA5"           "HABP2"          
## [277] "GCLM"            "FBXO38-DT"       "HERPUD1"         "IL23A"          
## [281] "TIAM2"           "TM6SF1"          "DLL1"            "LINC01694"      
## [285] "ADGRB1"          "SLC2A12"         "ENSG00000102543" "LINC00659"      
## [289] "TLR3"            "TLR5"            "DGKG"            "ASNS"           
## [293] "NKX2-2"          "FAM43A"          "IL15RA"          "PTGER3"         
## [297] "ENSG00000204356" "CDKN1A"          "C7orf31"         "FAAH2"          
## [301] "TFE3"            "ABHD4"           "ENSG00000198752" "SLC48A1"        
## [305] "IRF7"            "HRH1"            "AMIGO2"          "MIR22HG"        
## [309] "LINC00511"       "ENSG00000243811" "AARD"            "LINC00239"      
## [313] "CILP2"           "PCK2"            "SMPDL3B"         "LINC00882"      
## [317] "DMRTA2"          "TNFSF9"          "UBXN8"           "PSAT1"          
## [321] "NPAS1"           "ENSG00000214770" "CECR2"           "ENSG00000291136"
## [325] "EFNA1"           "ENTPD1"          "SLC12A5-AS1"     "PLCL1"          
## [329] "CD55"            "PRG4"            "ENSG00000120071" "SV2B"           
## [333] "CREB3L3"         "MAGOH-DT"        "MIAT"            "REPS2"          
## [337] "ETFBKMT"         "YWHAH-AS1"       "KRT10-AS1"       "ENO1-AS1"       
## [341] "LINC00685"       "LINC01277"       "ISG20"           "PPFIBP2"        
## [345] "INCA1"           "TMOD1"           "ENSG00000291027" "ARRDC3"         
## [349] "SYT1"            "SLC1A5"          "SESN2"           "YPEL4"          
## [353] "ULBP1"           "HRK"             "KDSR-DT"         "PPP2R3A"        
## [357] "FRMD3"           "TCAF2"           "ENSG00000006634" "NOS1AP"         
## [361] "DRC3"            "RBM11"           "ENSG00000160703" "SNAI1"          
## [365] "POF1B"           "TTTY14"          "SMTNL2"          "NCF2"           
## [369] "ZNF880"          "NCAM1"           "TMEM200A"        "LY9"            
## [373] "LINC02265"       "IQUB"            "MT1F"            "DRAM1"          
## [377] "SMOX"            "CLIP2"           "CEBPB"           "ENSG00000226029"
## [381] "TRPC5OS"         "CEBPB-AS1"       "ENSG00000159496" "AMOT"           
## [385] "FKTN-AS1"        "LINC01307"       "DAB1"            "RASD1"          
## [389] "MET"             "SGIP1"           "PHOSPHO1"        "GABARAPL1"      
## [393] "NECTIN2"         "IL7R"            "CCER2"           "FUT1"           
## [397] "WNT10A"          "ZNF516-AS1"      "ENSG00000139800" "LACTB2-AS1"     
## [401] "ENSG00000289115" "LINC02377"       "MYH14"           "ZBED3-AS1"      
## [405] "NIBAN1"          "TMEM217"         "PLAU"            "ENSG00000214046"
## [409] "MYOM2"           "DEPTOR"          "PMEL"            "FLRT1"          
## [413] "SMAD7"           "ADGRL3"          "PRKG2"           "ENSG00000115355"
## [417] "GLS2"            "LDB3"            "PCDHB14"         "MPZ"            
## [421] "TMEM156"         "ENSG00000161016" "TRAF3IP2"        "TLL1"           
## [425] "MFAP4"           "MAFB"            "SLC30A1"         "TEX14"          
## [429] "LINC00029"       "ENSG00000119684" "KANK3"           "LPIN3"          
## [433] "THEMIS2"         "SSC5D"           "RPL26L1-AS1"     "PRRT3-AS1"      
## [437] "SHISA2"          "CBR3-AS1"        "FMN1"            "SCN3A"          
## [441] "LACC1"           "LPAR4"           "JDP2-AS1"        "ENSG00000172071"
## [445] "NDUFA4L2"        "TMEM74B"         "ENSG00000110063" "XKRX"           
## [449] "PRMT5-DT"        "CDC20B"          "HYDIN"           "ENSG00000124172"
## [453] "ENSG00000205643" "PCDHGB7"         "ENSG00000287808" "TBL1X"          
## [457] "ST8SIA6"         "SCN3B"           "RAD9B"           "ADGRE1"         
## [461] "STC2"            "TLR1"            "ENSG00000163794" "GAS1RR"         
## [465] "ITGB5"           "JDP2"            "ENSG00000149451" "NR2F2"          
## [469] "CRPPA"           "ERBB3"           "ENSG00000284308" "CDNF"           
## [473] "ANXA3"           "GBP2"            "CALCRL"          "ENSG00000135436"
## [477] "TNFRSF11A"       "ZNF703"          "CHRNB4"          "VLDLR-AS1"      
## [481] "ENSG00000227500" "ADM2"            "ENSG00000180611" "LINC00992"      
## [485] "HSPA12B"         "UBQLN1-AS1"      "TRPC1"           "LMX1B"          
## [489] "PTGES"           "REXO5"           "CATSPERG"        "ENSG00000157992"
## [493] "LINC02341"       "LINC02018"       "PCDHGB6"         "NQO1-DT"        
## [497] "CREB5"           "TEX19"           "TGFBR3"          "SLC45A1"        
## [501] "LINC00648"       "COLGALT2"        "ENSG00000158406" "SYT11"          
## [505] "SH2D6"           "PCDH12"          "RPS6KA2"         "CST7"           
## [509] "ENSG00000198888" "DUSP8"           "ECI2-DT"         "TREML2"         
## [513] "ERICH2-DT"       "WDR93"           "PIFO"            "CDH15"          
## [517] "TMIE"            "HSPA1B"          "CEACAM1"         "SLC16A6"        
## [521] "CCDC148"         "ZNF35"           "ZFPM2"           "CFTR"           
## [525] "PCDH19"          "SP140"           "GPAT3"           "PPP1R15A"       
## [529] "FBXO39"          "CXCL3"           "TRAT1"           "SLC3A2"         
## [533] "SORBS1"          "MTTP"            "KIF17"           "ADTRP"          
## [537] "ZBTB46"          "RGS16"           "PIWIL4"          "FLRT3"          
## [541] "JAKMIP2"         "TMEM140"         "SPTA1"           "CSTA"           
## [545] "TC2N"            "SCN4B"           "PLAC1"           "JAKMIP3"        
## [549] "FOXA3"           "ENSG00000105516" "PRDM1"           "HTR2B"          
## [553] "F2RL2"           "MDN1-AS1"        "NQO1"            "SOX6"           
## [557] "ENSG00000254510" "PATJ"            "CHAC1"           "LINC02321"      
## [561] "HIPK1-AS1"       "GPR18"           "ENSG00000196132" "FAM166B"        
## [565] "CRB3"            "MKX"             "TLR6"            "SCN4A"          
## [569] "CLCA1"           "LY96"            "ENSG00000188739" "IL31RA"         
## [573] "NYAP1"           "PRPF19-DT"       "DPP4"            "TRPM6"          
## [577] "CCPG1"           "AKR1C3"          "TRPS1"           "CEACAM21"       
## [581] "IL12A"           "ID2"             "OLAH"            "MAP3K8"         
## [585] "ENSG00000171735" "GLIPR1"          "TRIB3"           "STYK1"          
## [589] "PCDH10-DT"       "MATN4"           "ENSG00000219200" "BEST1"          
## [593] "CCDC110"         "TSC22D3"         "KCNN3"           "FAM133A"        
## [597] "ENSG00000132005" "CLU"             "PDE11A"          "ADRA1A"         
## [601] "JUN"             "CHRNA6"          "DDIT3"           "LRRC2"          
## [605] "HEPH"            "ILDR1"           "LINC00632"       "ROCK1P1"        
## [609] "SATL1"           "SLC26A4-AS1"     "ENSG00000263528" "LUCAT1"         
## [613] "ENSG00000272010" "ENSG00000259905" "GPR132"          "FOSL1"          
## [617] "XKR3"            "SLC6A9"          "INPP5J"          "GPX2"           
## [621] "AKR1C8"          "ENSG00000163154" "CRIM1"           "UEVLD"          
## [625] "LINC02561"       "STX5-DT"         "SLC7A11"         "LINC02539"      
## [629] "TSPAN19"         "L3MBTL2-AS1"     "CYP19A1"         "PANX2"          
## [633] "CD80"            "BMP6"            "AKR1C2"          "OSGIN1"         
## [637] "MMP8"            "EGF"             "ENSG00000290683" "LINC00365"      
## [641] "HRG"             "GDAP1L1"         "ABCC3"           "UCP1"           
## [645] "HSPA6"           "RHOB"            "NMRAL2P"         "RASSF6"         
## [649] "HMOX1"           "C4orf17"

Now inspecting the gene biotypes

Let’s inspect the top up and down genes deeper !

Pathway enrichment analysis (PEA)

Gene Ontology (GO)

NOTE:

For the downstream analysis, only protein_coding genes will be kept to ensure the significance of the resulting terms!

Results

The top 30 most significant pathways enriched with these DEGs are

##  [1] "leukocyte activation"                        
##  [2] "lymphocyte activation"                       
##  [3] "multicellular organismal-level homeostasis"  
##  [4] "regulation of cell activation"               
##  [5] "regulation of leukocyte activation"          
##  [6] "cell-cell adhesion"                          
##  [7] "regulation of leukocyte differentiation"     
##  [8] "T cell activation"                           
##  [9] "myeloid leukocyte activation"                
## [10] "blood vessel morphogenesis"                  
## [11] "regulation of hemopoiesis"                   
## [12] "response to endoplasmic reticulum stress"    
## [13] "vasculature development"                     
## [14] "response to lipid"                           
## [15] "negative regulation of immune system process"
## [16] "regulation of T cell mediated immunity"      
## [17] "response to ketone"                          
## [18] "leukocyte differentiation"                   
## [19] "integrated stress response signaling"        
## [20] "epithelial cell proliferation"               
## [21] "blood vessel development"                    
## [22] "circulatory system process"                  
## [23] "response to organic cyclic compound"         
## [24] "mononuclear cell differentiation"            
## [25] "lymphocyte differentiation"                  
## [26] "endothelial cell migration"                  
## [27] "response to topologically incorrect protein" 
## [28] "angiogenesis"                                
## [29] "regulation of lymphocyte differentiation"    
## [30] "secretion"

The top 30 pathway with the highest RichFactor (Count per pathway length ratio)

##  [1] "double-strand break repair via break-induced replication"                    
##  [2] "regulation of ferroptosis"                                                   
##  [3] "regulation of heat generation"                                               
##  [4] "detection of molecule of bacterial origin"                                   
##  [5] "ferroptosis"                                                                 
##  [6] "DNA strand elongation involved in DNA replication"                           
##  [7] "epithelial cell fate commitment"                                             
##  [8] "regulation of IRE1-mediated unfolded protein response"                       
##  [9] "regulation of DNA-templated DNA replication initiation"                      
## [10] "negative regulation of endoplasmic reticulum unfolded protein response"      
## [11] "heat generation"                                                             
## [12] "negative regulation of leukocyte chemotaxis"                                 
## [13] "melanin biosynthetic process"                                                
## [14] "melanin metabolic process"                                                   
## [15] "secondary metabolite biosynthetic process"                                   
## [16] "integrated stress response signaling"                                        
## [17] "regulation of T cell mediated cytotoxicity"                                  
## [18] "secondary metabolic process"                                                 
## [19] "leukocyte activation involved in inflammatory response"                      
## [20] "negative regulation of chemotaxis"                                           
## [21] "negative regulation of leukocyte migration"                                  
## [22] "T cell mediated cytotoxicity"                                                
## [23] "regulation of T cell mediated immunity"                                      
## [24] "toll-like receptor signaling pathway"                                        
## [25] "endoplasmic reticulum unfolded protein response"                             
## [26] "female gonad development"                                                    
## [27] "development of primary female sexual characteristics"                        
## [28] "response to unfolded protein"                                                
## [29] "macrophage activation"                                                       
## [30] "positive regulation of tumor necrosis factor superfamily cytokine production"

Dotplot

Cnet plot

Gene Set Enrichment Analysis (GSEA)

GSEA is a functional class scoring method for enrichment analysis! It uses the whole set of genes ranked by the chosen scoring method: in our case, ranked by p.adjust and logFC values

• Higher scores represent the most significant Up regulated genes
• Lower scores represent the most significant Down regulated genes
• In between are the not significant genes

ranked_genes <- bitr(all_results$ensembl, 
                     fromType = "ENSEMBL",
                     toType = "ENTREZID",
                     OrgDb = "org.Hs.eg.db") %>% 
  dplyr::mutate(ensembl = ENSEMBL) %>% 
  inner_join(all_results, by = "ensembl") %>% 
  dplyr::select(ENSEMBL, ENTREZID, Gene_name, padj, log2FoldChange, Gene_biotype) %>% 
  dplyr::filter(Gene_biotype == "protein_coding",
                padj < 0.5,
                abs(log2FoldChange) >= 0.1) %>% 
  dplyr::mutate(padj = padj + 1e-300,
                rank = -log10(padj)*sign(log2FoldChange)) %>% 
  dplyr::group_by(ENTREZID) %>% 
  dplyr::slice_max(order_by = abs(rank), n = 1, with_ties = FALSE) %>% 
  ungroup() %>%
  dplyr::arrange(desc(rank))

ranked_genes <- setNames(ranked_genes$rank, ranked_genes$ENTREZID)
ranked_genes <- ranked_genes[intersect(names(ranked_genes), 
                                       keys(org.Hs.eg.db, keytype = "ENTREZID"))]

## [1] "No results found!"